This invention relates to astaxanthin. In one aspect, the invention relates to astaxanthin produced by yeast cells while in another aspect, the invention relates to methods of producing and cultivating mutant strains of Phaffia rhodozyma yeast cells that produce astaxanthin in excess of the typical Phaffia rhodozyma yeast cell found in nature. In yet another aspect, the invention relates to using products made from these yeast cells as a dietary supplement in various animal feeds.
A distinct red color is of prime importance to customer acceptance of certain food products, particularly aquatic food animals such as salmon, sea bream, trout, shrimp, lobster and many other marine animals. The oxygenated carotenoid astaxanthin (3,3xe2x80x2-dihydroxy-xcex2,xcex2-carotene-4,4xe2x80x2-dione) is responsible for the red color of these aquatic animals. In addition to being responsible for the characteristic color of these animals, astaxanthin plays a critical nutritional role in the life of these marine animals (Torrissen, 1989. Proc. Third Int. Symp. on Feeding and Nutr. in Fish, Toba August 28-September. 1, Japan, pp. 387-399, Meyers and Chen, 1982. World Aquaculture Society, Special Publication No. 3, pp. 153-165). These references are incorporated herein by reference. This carotenoid is also useful for adding pigmentation to the flesh and products of other animals, and to other foodstuffs, e.g. poultry and eggs, various dairy products, snack foods, and the like.
Astaxanthin is the most abundant carotenoid present in the aquatic world. Aquatic animals, like terrestrial animals, generally cannot synthesize astaxanthin or any other carotenoid, although many of these animals accumulate caroteniod compounds that are present in their diets. Some of these animals, such as crustaceans, can interconvert some carotenes to oxygenated forms of carotenoids (called xanthophylls) of which astaxanthin is the predominant compound formed. However, salmonid fishes and red sea bream accumulate dietary astaxanthin even though these fish cannot convert any other carotenoid compound to astaxanthin. Therefore, the astaxanthin present in salmonid and sea bream fish, and in products produced from these fish, must be derived directly from dietary sources.
Plants, including marine microalgae and special yeasts such as Phaffis rhodozyma, are the primary source of carotenoid compounds in the world. As noted above, carotenoids are not biosynthesized de novo by animals. However, animals in general require certain carotenoids from which they benefit directly or indirectly, and these carotenoids are obtained from dietary sources. Examples of substances essential to most animals that are derived from certain carotenoids are vitamin A and rhodopsin. In the marine world, animals that are low on the food chain, such as crustaceans, eat microalgae and other carotenoid containing organisms from the plant world, and convert the carotenoid compounds present in large part to astaxanthin by natural metabolic processes. The astaxanthin is then stored in the body of these astaxanthin producing animals.
Wild grown salmonid fishes and red sea bream obtain their astaxanthin from the crustaceans and other astaxanthin containing organisms that make up an important part of their diet. In the case of pen-grown salmonids and red sea bream, the feeds used to produce these fish must be supplemented with astaxanthin in order to provide a dietary source of this important natural constitutent of these fishes. Currently, synthetic astaxanthin is added to feeds prepared for production of salmonids and red sea bream in aquaculture to provide a source of this carotenoid compound. In some cases, synthetic canthaxanthin (an oxygenated carotenoid compound that is very closely related to astaxanthin) is used in place of astaxanthin in feeds for salmonids and red sea bream, but this compound does not function as well in these fishes as the naturally predominant astaxanthin.
Natural sources of dietary astaxanthin are in great demand by the aquacultural industries. Natural sources of dietary carotenoids that have been investigated for farmed fish include krill, crawfish, crustacean processing by-products, algae and higher plants. However, these natural sources tend to be too expensive and of limited availability and reliability to be commercially viable.
The red yeast, Phaffis rhodozyma, has received great attention from industry as a natural source of astaxanthin since it was isolated from tree sap, and the red color identified as astaxanthin (Miller, Yoneyama and Soneda. 1976. Int. J. Syst. Bacteriol. 26:286-291, Andrewes, Phaff and Starr. 1976. Phytochem. 15:1003-1007) Phaffia rhodozma was first demonstrated to pigment salmonid fishes in 1977 (Johnson, Conklin and Lewis. 1977. J. Fish. Res. Board. Can. 34:2417-2421, Johnson, Villa and Lewis. 1980. Aquaculture. 20:123-134). The potential advantages of Phaffia rhodozma as a source of carotenoid pigments for aquaculture are that it is a natural product rich in essential nutrients (e.g. protein, lipids and B-vitamins) and that it contains astaxanthin (Johnson, Villa and Lewis. 1980. Aquaculture. 20:123-134). However, natural isolates of Phaffia rhodozyma produce so little astaxanthin (typically 100 to 300 parts per million (ppm)) that they are not practical or economical pigment sources for aquaculture (Torrissen, Hardy and Shearer. 1989. Reviews in Aquatic Science 1:209-225, Johnson and An. 1991. CRC Crit. Rev. Biotech. 11:297-326). If Phaffia strains are to be an economically feasible feed additive for coloring aquatic animals, or any other potential foodstuff (animal or otherwise), then astaxanthin over-producing strains must be developed. Each of the references cited in this paragraph are incorporated herein by reference.
Mutants of naturally occurring xe2x80x9cwild-typexe2x80x9d Phaffia have been described in the literature, allegedly capable of generating higher levels of astaxanthin than the wild-type yeasts (International Publication No. WO 88/08025 International Application No. PCT/DK88/00068); EPO Publication No. 0 438 182 A1 (EPO Application No. 91900682.3); EPO Publication No. 0 454 024 A2 (EPO Application No. 91106436.8); International Publication No. WO 91/02060 (International Application No. PCT/US90/00558); EPO Publication No. 0 474 347 A1 (EPO Application No. 91306489.5); and EPO Publication No. 0 427 405 A1 (EPO Application No. 90311254.8), all of which are incorporated herein by reference). These strains reportedly produce higher levels of astaxanthin than the wild-type isolates under specific conditions. However, these mutant strains produce higher levels of astaxanthin only at relatively low biomass concentrations. At relatively high biomass concentrations, these mutant strains produce only low levels of astaxanthin which are not high enough to be practical.
Thus far, none of the reported Phaffia strains are capable of producing astaxanthin efficiently enough to compete economically with synthetic astaxanthin. Commercially viable strains have to produce astaxanthin at substantially higher levels than strains reported in the literature. To develop an economically viable astaxanthin production process, a strain should produce in excess of 3,000 ppm, preferably in excess of 4,000 ppm, astaxanthin based on dry yeast solids at more than 4 wt %, preferably more than 6 wt % dry yeast solids (dys) in a large volume of nutrient medium, e.g. 1,500 liters (1) or more. As here used, xe2x80x9cwt % dry yeast solidsxe2x80x9d or simply xe2x80x9cdry yeast solidsxe2x80x9d are washed solids determined by the method described in Official Method of Analysis, A.O.A.C. 14th Edition (1984) Sections 10.215-10.225, which is incorporated herein by reference.
Besides the need to develop a suitable strain of Phaffia rhodozma for commercial astaxanthin production, methods for cultivating Phaffia rhodozma also need to be developed which maximize astaxanthin production in large fermentors. Only limited literature sources are available dealing with the growth of Phaffia and production processes of astaxanthin in large fermentors (International Publication No. WO 88/08025 (International Application No. PCT/DK88/00068), and EPO Publication No. 0 454 024 A2 (EPO Application No. 91106436.8) both of which are incorporated herein by reference). These reported fermentation processes give significantly lower yeast solids and astaxanthin levels than that required for an economically viable commercial scale fermentation process.
Not only do the various elements of producing astaxanthin from yeast cells require improvement, but so do the elements of astaxanthin absorption and deposition in animal flesh and animal products. Carotenoid absorption and deposition in fish are affected by various factors: genetics, size, age, sex, duration of pigment feeding, environmental factors, and diet composition (Torrissen, Hardy and Shearer: 1989: Reviews in Aquatic Science, 1:209-225, which is incorporated herein by reference).
Various reports are available concerning the formulation of Phaffia or carotenoid products (International Publication No. WO 88/08025 (International Application No. PCT/DK88/00068), EPO Publication No. 0 474 347 A1 (EPO Application No. 91306489.5), EPO Publication No. 0 454 024 A2 (EPO Application No. 91106436.8), and Japanese Patent Applications 57-206342, 2238855 and 90 JP-285090 are exemplary, all of which are incorporated herein by reference). In these reports, Phaffia products were formulated in order to protect the pigments during drying as well as during subsequent processing into fish feed. Various antioxidants and stabilizers were added to Phaffia preparations for those purposes.
The body temperature of salmonid fishes is equal to the temperature of the water in which they inhabit, e.g. generally 0 to 14 degrees Centigrade (C). This means that the body temperature of these fishes can be, and occasionally is, lower than 10 C. Astaxanthin in Phaffia is concentrated in oil droplets . Phaffia oil contains about 13% palmitic acid (16:00) with a melting point of 64 C, and about 32% oleic acid (18:1n9) with a melting point of 16 C. Because of these high melting point fatty acids, Phaffia oil solidifies near 10 C. This makes it difficult for fishes to incorporate Phaffia astaxanthin at water temperatures below 10 C at which Phaffia oil is solidified.
In one embodiment of the present invention, we have developed and isolated novel strains of Phaffia rhodozyma which produce greater than 3,000 ppm, often more than 4,000 ppm, astaxanthin based on dry yeast solids at more than 4 wt %, often more than 6 wt %, dry yeast solids when grown under suitable conditions and in a working volume of nutrient media of at least about 1,500 1. These Phaffia strains can produce astaxanthin economically on a commercial production scale for the aquacultural and food industries. Certain of these Phaffia strains were deposited with the American Type Culture Collection of Rockville, Md., on Apr. 6, 1993 under numbers ATCC-74218 (UBV-AX1), ATCC-74219 (UBV-AX2), ATCC-74220 (UBV-AX3), and ATCC-74221 (UBV-AX4).
In another embodiment of this invention, we have discovered methods for the cultivation of Phaffia rhodozyma cells which produce high yeast solids and high levels of astaxanthin. Generally, these methods include providing special fermentation conditions that promote the growth of the organism in terms of an increase in cell numbers during a growth phase, and in terms of an enhanced and steady accumulation of astaxanthin in the cells during a maturation phase. More specifically, these methods include exposing the yeast cells during the growth and maturation phases of fermentation to a source of light, and/or extending and stimulating the astaxanthin formation during the maturation phase by controlling the feed rate of fermentable sugar, e.g. glucose, or by providing the yeast cells with energy sources that are slowly metabolized, e.g. glycerol.
In another embodiment of this invention, we have discovered that the Phaffia strains of this invention need not be disrupted to make astaxanthin available to salmonids, sea bream, crustaceans, and other animals. Astaxanthin in undisrupted cells is more stable during drying, storage and feed preparation processes.
In yet another embodiment of this invention, we have discovered that a novel formulation of Phaffia product increases the astaxanthin deposition in fishes. This formulation is a blend of undisrupted Phaffia cells, vegetable oil or a blend of vegetable oils with a low solidification point, an emulsifier, and an antioxidant.